The objective of this grant proposal is to develop and make available to the community tools to identify genes responsible for any phenotype that can be measured in the model filamentous fungus, Neurospora crassa. We propose to develop as a community resource two large mapping populations, which will enable the identification of quantitative trait loci (QTLs) controlling naturally varying complex phenotypes. One of our populations comprises 500 wild isolates from a single phylogenetic clade of N. crassa, to be used for association mapping, the method of choice for identification of loci of small effect. The other population is an advanced intercross between two divergent N. crassa strains, to allow linkage mapping of rare alleles at high genetic resolution. For each population, we will measure genotype and gene expression via massively parallel signature sequencing using the Solexa platform. These resources will enable any research lab to score the strains for a trait of interest and map the underlying QTLs. As a proof of principle, we will characterize the wild and advanced intercross strains with respect to phenotypes that have broad interest to the Neurospora research community: asexual spore reproduction, sexual reproduction, vegetative growth, hyphal architecture and cooperative behavior during spore germination. We will pioneer the use of the mRNA expression data from our Solexa experiment to identify the molecular players underlying these macroscopic traits, as the expression of genes is shown to co-vary with traits across individuals. In addition, the expression measurements will be used to detail the N. crassa regulatory network, as we analyze covariation between expression levels and genetically map their controlling regulators. Our specific aims are (i) to construct the advanced intercross and characterize phenotypes that vary among these strains and the wild isolates;(ii) to perform short-read sequencing on cDNAs using the Solexa platform from each individual in each mapping population, to generate dense genotyping data and measure gene expression;and (iii) to conduct linkage and association mapping of mRNA expression and our proof-of-principle organismal traits. We believe that the mapping between genotypes, phenotypes, and gene expression levels that we propose would propel N. crassa to the forefront as a model for the study of natural variation in all eukaryotic species. Relevance to Public Health: Using N. crassa, we propose to study genetic differences between individuals that control traits like growth, organism shape, and sensitivity to drugs. N. crassa is a model for pathogenic fungi, and we aim to understand many aspects of its biology that are important for disease and treatment. Also, since so little is known about the 'principles of -genetic differences between individuals, our work will provide hypotheses about the genetics of other species, like humans.